SRM/MRM Assay for the Fibroblast Growth Factor Receptor 2 (FGFR2) protein

ABSTRACT

Methods are provided for quantifying the fibroblast growth factor receptor 2 protein (FGFR2) directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) mass spectrometry. The biological sample may be selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells. A protein sample is prepared from the biological sample and the FGFR2 protein is quantitated in the sample using the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one fragment peptide derived from FGFR2.

This application claims priority to provisional application Ser. No.62/161,757, filed May 14, 2015, the contents of which are herebyincorporated by reference in their entirety. This application alsocontains a sequence listing submitted electronically via EFS-web, whichserves as both the paper copy and the computer readable form (CRF) andconsists of a file entitled “001152_8045_US01_SEQ_LISTING”, which wascreated on May 13, 2016, which is 577 bytes in size, and which is alsoincorporated by reference in its entirety.

INTRODUCTION

Cancer is treated with a collection of therapeutic agents that killgrowing and dividing cells and that function in a variety of ways. Acommon collection of chemotherapeutic agents has been used for decades,either individually or in combinations, and this common collection ofagents has become the traditional and routine cancer treatment inclinical oncology practice. These traditional chemotherapeutics agentsact by killing all cells that divide rapidly, one of the main propertiesof most cancer cells. However, these agents also kill growing normalcells and thus these agents are not considered to be “targeted”approaches to killing cancer cells. In recent years a large group ofcancer therapeutic agents has been developed that target only cancercells where the therapeutic agent specifically attacks a protein that isonly expressed by the cancer cells and not by normal cells. Thisapproach is considered to be a “targeted” approach to cancer therapy.Most recently, another approach to killing cancer cells in a “targeted”fashion has been to specifically modulate the immune system to enhancethe ability of the cancer patient's immune system to kill cancer cells.

Therapeutic agents that target the fibroblast growth factor receptor 2protein, which is also referred to as FGFR2, have shown promise in earlyclinical trials. However, only those patients whose cancer cells expresshigh amounts of the FGFR2 protein are likely to benefit from treatmentwith such FGFR2-targeted therapeutic agents. The methods described belowprovide a quantitative proteomics-based assay that delivers a relevantmeasure of activation of the FGFR2 signal pathway as FGFR2 is notnormally expressed in normal tissue and/or normal epithelial cells. Inparticular, the methods provide a mass spectrometry assay thatquantifies FGFR2 in formalin fixed tissues from cancer patients and thatenables improved treatment decisions for cancer therapy.

Specific peptides derived from subsequences of the fibroblast growthfactor receptor 2 protein, also referred to as CD332, Keratinocytegrowth factor receptor, and KGFR, and referred to herein as FGFR2, areprovided. The peptide sequence and fragmentation/transition ions foreach peptide are particularly useful in a mass spectrometry-basedSelected Reaction Monitoring (SRM), which can also be referred to as aMultiple Reaction Monitoring (MRM) assay, and is referred to herein asSRM/MRM. The use of peptides for SRM/MRM quantitative analysis of theFGFR2 protein is described.

This SRM/MRM assay can be used to measure relative or absolutequantitative levels of one or more of the specific peptides from theFGFR2 protein and therefore provide a mass spectrometric methods ofmeasuring the amount of the FGFR2 protein in a given protein preparationobtained from a biological sample.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by references in their entirety. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue reagents and protocol available from Expression Pathology Inc.(Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatients tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue by far themost common method of preserving cancer tissue samples worldwide and isthe accepted convention for standard pathology practice. Aqueoussolutions of formaldehyde are referred to as formalin. “100%” formalinconsists of a saturated solution of formaldehyde (about 40% by volume or37% by mass) in water, with a small amount of stabilizer, usuallymethanol to limit oxidation and degree of polymerization. The mostcommon way in which tissue is preserved is to soak whole tissue forextended periods of time (8 hours to 48 hours) in aqueous formaldehyde,commonly termed 10% neutral buffered formalin, followed by embedding thefixed whole tissue in paraffin wax for long term storage at roomtemperature. Thus molecular analytical methods to analyze formalin fixedcancer tissue will be the most accepted and heavily utilized methods foranalysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate andprecise quantitative levels of the FGFR2 protein within the specifictissue samples (e.g., cancer tissue sample) of the patient or subjectfrom whom the tissue (biological sample) was collected and preserved.This not only provides diagnostic and prognostic information about thecancer, but also permits a physician or other medical professional tomore accurately determine appropriate therapy for the patient. Such anassay that provides diagnostically, prognostically, and therapeuticallyimportant information about levels of protein expression in a diseasedtissue or other patient sample is termed a companion diagnostic assay.For example, such an assay can be designed to diagnose the stage ordegree of a cancer and determine a therapeutic agent to which a patientis most likely to respond.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the FGFR2 protein and also can measureabsolute or relative levels of specific modified peptides from the FGFR2protein. Examples of modifications include phosphorylated amino acidresidues and glycosylated amino acid residues that may be present on thepeptides.

Relative quantitative levels of the FGFR2 protein are determined by theSRM/MRM methodology for example by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual FGFR2 peptide in different samples.

Alternatively, it is possible to compare multiple SRM/MRM signature peakareas for multiple FGFR2 signature peptides, where each peptide has itsown specific SRM/MRM signature peak, to determine the relative FGFR2protein content in one biological sample with the FGFR2 protein contentin one or more additional or different biological samples. In this way,the amount of a particular peptide, or peptides, from the FGFR2 protein,and therefore the amount of the FGFR2 protein, is determined relative tothe same FGFR2 peptide, or peptides, across 2 or more biological samplesunder the same experimental conditions. In addition, relativequantitation can be determined for a given peptide, or peptides, fromthe FGFR2 protein within a single sample by comparing the signature peakarea for that peptide by SRM/MRM methodology to the signature peak areafor another and different peptide, or peptides, from a differentprotein, or proteins, within the same protein preparation from thebiological sample. In this way, the amount of a particular peptide fromthe FGFR2 protein, and therefore the amount of the FGFR2 protein, isdetermined relative one to another within the same sample. Theseapproaches permit quantitation of an individual peptide, or peptides,from the FGFR2 protein to the amount of another peptide, or peptides,between samples and within samples wherein the amounts as determined bysignature peak area are relative one to another, regardless of theabsolute weight to volume or weight to weight amounts of the FGFR2peptide in the protein preparation from the biological sample. Relativequantitative data about individual signature peak areas betweendifferent samples are normalized to the amount of protein analyzed persample. Relative quantitation can be performed across many peptides frommultiple proteins and the FGFR2 protein simultaneously in a singlesample and/or across many samples to gain insight into relative proteinamounts of one peptide/protein with respect to other peptides/proteins.

Absolute quantitative levels of the FGFR2 protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the FGFR2 protein in one biological sampleis compared to the SRM/MRM signature peak area of a spiked internalstandard. In one embodiment, the internal standard is a syntheticversion of the same exact FGFR2 peptide that contains one or more aminoacid residues labeled with one or more heavy isotopes. Such an isotopelabeled internal standard is synthesized so that when analyzed by massspectrometry it generates a predictable and consistent SRM/MRM signaturepeak that is different and distinct from the native FGFR2 peptidesignature peak and which can be used as a comparator peak. Thus when theinternal standard is spiked into a protein preparation from a biologicalsample in known amounts and analyzed by mass spectrometry, the SRM/MRMsignature peak area of the native peptide is compared to the SRM/MRMsignature peak area of the internal standard peptide, and this numericalcomparison indicates either the absolute molarity and/or absolute weightof the native peptide present in the original protein preparation fromthe biological sample. Absolute quantitative data for fragment peptidesare displayed according to the amount of protein analyzed per sample.Absolute quantitation can be performed across many peptides, and thusproteins, simultaneously in a single sample and/or across many samplesto gain insight into absolute protein amounts in individual biologicalsamples and in entire cohorts of individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, and/or the patient prognosis, for example, directly inpatient-derived tissue, such as formalin fixed tissue, and to aid indetermining which therapeutic agent would be most advantageous for usein treating that patient. Cancer tissue that is removed from a patienteither through surgery, such as for therapeutic removal of partial orentire tumors, or through biopsy procedures conducted to determine thepresence or absence of suspected disease, is analyzed to determinewhether or not a specific protein, or proteins, and which forms ofproteins, are present in that patient tissue. Moreover, the expressionlevel of a protein, or multiple proteins, can be determined and comparedto a “normal” or reference level found in healthy tissue. Normal orreference levels of proteins found in healthy tissue may be derivedfrom, for example, the relevant tissues of one or more individuals thatdo not have cancer. Alternatively, normal or reference levels may beobtained for individuals with cancer by analysis of relevant tissues notaffected by the cancer. Assays of protein levels (e.g., FGFR2 levels)can also be used to diagnose, and provide prognostic information about,the stage of cancer in a patient or subject diagnosed with cancer byemploying the FGFR2 levels. The level of an individual FGFR2 peptide isdefined as the molar amount of the peptide determined by the SRM/MRMassay per total amount of protein lysate analyzed. Information regardingFGFR2 can thus be used to aid in determining the stage or grade of acancer by correlating the level of the FGFR2 protein (or fragmentpeptides of the FGFR2 protein) with levels observed in normal tissues.Once the quantitative amount of the FGFR2 protein has been determined inthe cancer cells, that information can be matched to a list oftherapeutic agents (chemical and biological) developed to specificallytreat cancer tissue that is characterized by, for example, abnormalexpression of the protein or protein(s) (e.g., FGFR2) that were assayed.Matching information from an FGFR2 protein assay to a list oftherapeutic agents that specifically targets, for example, the FGFR2protein or cells/tissue expressing the protein, defines what has beentermed a personalized medicine approach to treating disease. The assaymethods described herein form the foundation of a personalized medicineapproach by using analysis of proteins from the patient's own tissue asa source for diagnostic and treatment decisions.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from the FGFR2 protein,prepared for example by digesting with a protease of known specificity(e.g. trypsin), can be used as a surrogate reporter to determine theabundance of FGFR2 protein in a sample using a mass spectrometry-basedSRM/MRM assay. Similarly, any predicted peptide sequence containing anamino acid residue at a site that is known to be potentially modified inthe FGFR2 protein also might potentially be used to assay the extent ofmodification of the FGFR2 protein in a sample.

FGFR2 fragment peptides may be generated by a variety of methodsincluding by the use of the Liquid Tissue protocol provided in U.S. Pat.No. 7,473,532. The Liquid Tissue protocol and reagents are capable ofproducing peptide samples suitable for mass spectroscopic analysis fromformalin fixed paraffin embedded tissue by proteolytic digestion of theproteins in the tissue/biological sample. In the Liquid Tissue protocolthe tissue/biological is heated in a buffer for an extended period oftime (e.g., from about 80° C. to about 100° C. for a period of time fromabout 10 minutes to about 4 hours) to reverse or release proteincross-linking. The buffer employed is a neutral buffer, (e.g., aTris-based buffer, or a buffer containing a detergent). Following heattreatment the tissue/biological sample is treated with one or moreproteases, including but not limited to trypsin, chymotrypsin, pepsin,and endoproteinase Lys-C for a time sufficient to disrupt the tissue andcellular structure of said biological sample and to liquefy the sample(e.g. a period of time from 30 minutes to 24 hours at a temperature from37° to 65° C.). The result of the heating and proteolysis is a liquid,soluble, dilutable biomolecule lysate.

Surprisingly, it was found that many potential peptide sequences fromthe FGFR2 protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. This was especially true for peptides derived fromformalin-fixed tissue. As it was not possible to predict the mostsuitable peptides for MRM/SRM assay, it was necessary to experimentallyidentify modified and unmodified peptides in actual Liquid Tissuelysates to develop a reliable and accurate SRM/MRM assay for the FGFR2protein. While not wishing to be bound by any theory, it is believedthat some peptides might, for example, be difficult to detect by massspectrometry because they either do not ionize well or they producefragments that are not distinct from peptides derived from otherproteins. Peptides may also fail to resolve well in separation (e.g.,liquid chromatography), or may adhere to glass or plastic ware.

FGFR2 peptides found in various embodiments of this disclosure (e.g.,Table 1) were derived from the FGFR2 protein by protease digestion ofall the proteins within a complex Liquid Tissue lysate prepared fromcells procured from formalin fixed cancer tissue. Unless notedotherwise, in each instance the protease was trypsin. The Liquid Tissuelysate was then analyzed by mass spectrometry to determine thosepeptides derived from the FGFR2 protein that are detected and analyzedby mass spectrometry. Identification of a specific preferred subset ofpeptides for mass-spectrometric analysis is based on; 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue lysates, and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissuereagents and protocol that entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, as forexample including but not limited to the protease trypsin. Each proteinlysate is turned into a collection of peptides by digestion of intactpolypeptides with the protease. Each Liquid Tissue lysate was analyzed(e.g., by ion trap mass spectrometry) to perform multiple globalproteomic surveys of the peptides where the data was presented asidentification of as many peptides as could be identified by massspectrometry from all cellular proteins present in each protein lysate.An ion trap mass spectrometer or another form of a mass spectrometerthat is capable of performing global profiling for identification of asmany peptides as possible from a single complex protein/peptide lysateis typically employed. Ion trap mass spectrometers however may be thebest type of mass spectrometer for conducting global profiling ofpeptides. Although an SRM/MRM assay can be developed and performed onany type of mass spectrometer, including a MALDI, ion trap, or triplequadrupole, advantageously the instrument platform for an SRM/MRM assayis a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single MSanalysis of a single lysate under the conditions employed, then thatlist of peptides was collated and used to determine the proteins thatwere detected in that lysate. That process was repeated for multipleLiquid Tissue lysates, and the very large list of peptides was collatedinto a single dataset. That type of dataset can be considered torepresent the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the FGFR2 protein.

In one embodiment, the FGFR2 tryptic peptides identified as useful inthe determination of absolute or relative amounts of the FGFR2 proteininclude one or more of SEQ ID NO:1 and SEQ ID NO:2, both of which arelisted in Table 1. Each of these peptides was detected by massspectrometry in Liquid Tissue lysates prepared from formalin fixed,paraffin embedded tissue. Thus, each peptide is a candidate for use indeveloping a quantitative SRM/MRM assay for the FGFR2 protein in humanbiological samples, including directly in formalin fixed patient tissue.

TABLE 1 Mono Precursor Peptide Isotopic Charge Precursor Transition IonSEQ ID sequence Mass State m/z m/z Type SEQ ID NO: 1 EAVTVAVK  815.47522 408.745 317.218 y3 2 408.745 416.286 y4 2 408.745 517.334 y5 2 408.745616.402 y6 SEQ ID NO: 2 YGPDGLPY 1121.5756 2 561.795 451.753 y8 LK 2561.795 520.312 y4 2 561.795 690.418 y6 2 561.795 805.445 y7 2 561.795902.498 y8The FGFR2 tryptic peptides listed in Table 1 were detected from multipleLiquid Tissue lysates of multiple different formalin fixed tissues ofdifferent human organs including prostate, colon, and breast. Each ofthose peptides is considered useful for quantitative SRM/MRM assay ofthe FGFR2 protein in formalin fixed tissue. Further data analysis ofthese experiments indicated no preference is observed for either ofthese peptides from any specific organ site. Thus, each of thesepeptides is believed to be suitable for conducting SRM/MRM assays of theFGFR2 protein on a Liquid Tissue lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

An important consideration when conducting an SRM/MRM assay is the typeof instrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for an SRM/MRM assay ispresently most likely considered to be a triple quadrupole instrumentplatform. That type of a mass spectrometer is presently the mostsuitable instrument for putative analysis of a single isolated targetpeptide within a very complex protein lysate that may consist ofhundreds of thousands to millions of individual peptides from all theproteins contained within a cell.

In order to most efficiently implement an SRM/MRM assay for each peptidederived from the FGFR2 protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer) to perform thecorrect and focused analysis of specific targeted peptide(s), such thatthe assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific FGFR2 peptides, may include one or more of the mono isotopicmass of the peptide, its precursor charge state, the precursor m/zvalue, the m/z transition ions, and the ion type of each transition ion.Additional peptide information that may be used to develop an SRM/MRMassay for the FGFR2 protein is shown for these FGFR2 peptides from Table1.

The method described below was used to: 1) identify candidate peptidesfrom the FGFR2 protein that can be used for a mass spectrometry-basedSRM/MRM assay for the FGFR2 protein, 2) develop an individual SRM/MRMassay, or assays, for target peptides from the FGFR2 protein in order tocorrelate and 3) apply quantitative assays to cancer diagnosis and/orchoice of optimal therapy.

Assay Method

-   1. Identification of SRM/MRM candidate fragment peptides for the    FGFR2 protein    -   a. Prepare a Liquid Tissue protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the FGFR2 protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the FGFR2 protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length FGFR2 protein potentially can be        measured, but preferred peptides used for development of the        SRM/MRM assay are those that are identified by mass spectrometry        directly in a complex Liquid Tissue protein lysate prepared from        a formalin fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the        FGFR2 protein-   2. Mass Spectrometry Assay for Fragment Peptides from the FGFR2    Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue        lysate is applied to peptides from the FGFR2 protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the FGFR2 protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the protein in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                FGFR2 protein by comparing the SRM/MRM signature peak                area from a given FGFR2 peptide detected in a Liquid                Tissue lysate from one formalin fixed biological sample                to the same SRM/MRM signature peak area of the same                FGFR2 fragment peptide in at least a second, third,                fourth or more Liquid Tissue lysates from least a                second, third, fourth or more formalin fixed biological                samples            -   2. Determining increased or decreased presence of the                FGFR2 protein by comparing the SRM/MRM signature peak                area from a given FGFR2 peptide detected in a Liquid                Tissue lysate from one formalin fixed biological sample                to SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                FGFR2 protein by comparing the SRM/MRM signature peak                area for a given FGFR2 peptide to the SRM/MRM signature                peak areas from other fragment peptides derived from                different proteins within the same Liquid Tissue lysate                from the formalin fixed biological sample in order to                normalize changing levels of FGFR2 protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the FGFR2 protein, where the modifications include but                are not limited to phosphorylation and/or glycosylation,                and where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the FGFR2 protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the FGFR2 protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.-   3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and    Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the FGFR2 protein and demonstrate that the        previously-determined association, as well understood in the        field of cancer, of FGFR2 protein expression to the        stage/grade/status of cancer in patient tumor tissue is        confirmed    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the FGFR2 protein and demonstrate correlation        with clinical outcomes from different treatment strategies,        wherein this correlation has already been demonstrated in the        field or can be demonstrated in the future through correlation        studies across cohorts of patients and tissue from those        patients. Once either previously established correlations or        correlations derived in the future are confirmed by this assay        then the assay method can be used to determine optimal treatment        strategy

Specific and unique characteristics about specific FGFR2 peptides weredeveloped by analysis of all FGFR2 peptides on both an ion trap andtriple quadrupole mass spectrometers. That information includes themonoisotopic mass of the peptide, its precursor charge state, theprecursor m/z value, the transition m/z values of the precursor, and theion types of each of the identified transitions. That information mustbe determined experimentally for each and every candidate SRM/MRMpeptide directly in Liquid Tissue lysates from formalin fixedsamples/tissue; because, interestingly, not all peptides from the FGFR2protein can be detected in such lysates using SRM/MRM as describedherein, indicating that FGFR2 peptides not detected cannot be consideredcandidate peptides for developing an SRM/MRM assay for use inquantitating peptides/proteins directly in Liquid Tissue lysates fromformalin fixed samples/tissue.

A particular SRM/MRM assay for a specific FGFR2 peptide is performed ona triple quadrupole mass spectrometer. An experimental sample analyzedby a particular FGFR2 SRM/MRM assay is for example a Liquid Tissueprotein lysate prepared from a tissue that had been formalin fixed andparaffin embedded. Data from such as assay indicates the presence of theunique SRM/MRM signature peak for this FGFR2 peptide in the formalinfixed sample.

Specific transition ion characteristics for this peptide are used toquantitatively measure a particular FGFR2 peptide in formalin fixedbiological samples. These data indicate absolute amounts of this FGFR2peptide as a function of molar amount of the peptide per microgram ofprotein lysate analyzed. Assessment of FGFR2 protein levels in tissuesbased on analysis of formalin fixed patient-derived tissue can providediagnostic, prognostic, and therapeutically-relevant information abouteach particular patient. In one embodiment, this disclosure describes amethod for measuring the level of the fibroblast growth factor receptor2 protein (FGFR2) in a biological sample, comprising detecting and/orquantifying the amount of one or more modified or unmodified FGFR2fragment peptides in a protein digest prepared from said biologicalsample using mass spectrometry; and calculating the level of modified orunmodified FGFR2 protein in said sample; and wherein said level is arelative level or an absolute level. In a related embodiment,quantifying one or more FGFR2 fragment peptides comprises determiningthe amount of the each of the FGFR2 fragment peptides in a biologicalsample by comparison to an added internal standard peptide of knownamount, wherein each of the FGFR2 fragment peptides in the biologicalsample is compared to an internal standard peptide having the same aminoacid sequence. In some embodiments the internal standard is anisotopically labeled internal standard peptide comprises one or moreheavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H orcombinations thereof.

The method for measuring the level of the FGFR2 protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) maybe used as a diagnostic and/or prognostic indicator of cancer in apatient or subject. In one embodiment, the results from measurements ofthe level of the FGFR2 protein may be employed to determine thediagnostic stage/grade/status of a cancer by correlating (e.g.,comparing) the level of FGFR2 protein found in a tissue with the levelof that protein found in normal and/or cancerous or precanceroustissues.

Because both nucleic acids and protein can be analyzed from the sameLiquid Tissue™ biomolecular preparation it is possible to generateadditional information about disease diagnosis and drug treatmentdecisions from the nucleic acids in same sample upon which proteins wereanalyzed. For example, if the FGFR2 protein is expressed by certaincells at increased levels, when assayed by SRM the data can provideinformation about the state of the cells and their potential foruncontrolled growth, potential drug resistance and the development ofcancers can be obtained. At the same time, information about the statusof the FGFR2 genes and/or the nucleic acids and proteins they encode(e.g., mRNA molecules and their expression levels or splice variations)can be obtained from nucleic acids present in the same Liquid Tissue™biomolecular preparation can be assessed simultaneously to the SRManalysis of the FGFR2 protein. Any gene and/or nucleic acid not from theFGFR2 and which is present in the same biomolecular preparation can beassessed simultaneously to the SRM analysis of the FGFR2 protein. In oneembodiment, information about the FGFR2 protein and/or one, two, three,four or more additional proteins may be assessed by examining thenucleic acids encoding those proteins. Those nucleic acids can beexamined, for example, by one or more, two or more, or three or more of:sequencing methods, polymerase chain reaction methods, restrictionfragment polymorphism analysis, identification of deletions, insertions,and/or determinations of the presence of mutations, including but notlimited to, single base pair polymorphisms, transitions, transversions,or combinations thereof.

1. A method for measuring the level of the fibroblast growth factorreceptor 2 protein (FGFR2) in a biological sample, comprising detectingand/or quantifying the amount of one or more modified or unmodifiedFGFR2 fragment peptides in a protein digest prepared from saidbiological sample using mass spectrometry; and calculating the level ofmodified or unmodified FGFR2 protein in said sample; and wherein saidlevel is a relative level or an absolute level.
 2. The method of claim1, further comprising the step of fractionating said protein digestprior to detecting and/or quantifying the amount of one or more modifiedor unmodified FGFR2 fragment peptides.
 3. The method of claim 2, whereinsaid fractionating step is selected from the group consisting of gelelectrophoresis, liquid chromatography, capillary electrophoresis,nano-reversed phase liquid chromatography, high performance liquidchromatography, or reverse phase high performance liquid chromatography.4. The method of claim 1, wherein said protein digest comprises aprotease digest.
 5. The method of claim 4, wherein said protein digestcomprises a trypsin digest.
 6. The method of claim 1, wherein the FGFR2fragment peptide comprises an amino acid sequence as set forth in SEQ IDNO:1 or SEQ ID NO:2.
 7. The method of claim 1, wherein the tissue isformalin fixed tissue.
 8. The method of claim 7, wherein the tissue isparaffin embedded tissue.
 9. The method of claim 8, wherein the tissueis obtained from a tumor.
 10. The method of claim 1, further comprisingquantifying a modified or unmodified FGFR2 fragment peptide.
 11. Themethod of claim 10, wherein quantifying the FGFR2 fragment peptidecomprises comparing an amount of one or more FGFR2 fragment peptideshaving the sequence of SEQ ID NO:1 or SEQ ID NO:2 in one biologicalsample to the amount of the same FGFR2 fragment peptide or peptides in adifferent and separate biological sample.
 12. The method of claim 1,wherein quantifying one or more FGFR2 fragment peptides comprisesdetermining the amount of an FGFR2 fragment peptide or peptides in abiological sample by comparison to an added internal standard peptide ofknown amount, wherein each FGFR2 fragment peptide or peptides in thebiological sample is compared to an internal standard peptide having thesame amino acid sequence.
 13. The method of claim 12, wherein theinternal standard peptide is an isotopically labeled peptide.
 14. Themethod of claim 13, wherein the isotopically labeled internal standardpeptide comprises one or more heavy stable isotopes selected from ¹⁸O,¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.
 15. The method of claim1, wherein detecting and/or quantifying the amount of one or moremodified or unmodified FGFR2 fragment peptides in the protein digestindicates the presence of modified or unmodified FGFR2 protein and anassociation with cancer in the subject.
 16. The method of claim 15,further comprising correlating the results of said detecting and/orquantifying the amount of one or more modified or unmodified FGFR2fragment peptides, or the level of said FGFR2 protein to the diagnosticstage/grade/status of the cancer.
 17. The method of claim 16, whereincorrelating the results of said detecting and/or quantifying the amountof one or more modified or unmodified FGFR2 fragment peptides, or thelevel of said FGFR2 protein to the diagnostic stage/grade/status of thecancer is combined with detecting and/or quantifying the amount of otherproteins or peptides from other proteins in a multiplex format toprovide additional information about the diagnostic stage/grade/statusof the cancer.
 18. The method of claim 16, further comprising selectingfor the subject from which said biological sample was obtained atreatment based on the presence, absence, or amount of one or more FGFR2fragment peptides or the level of FGFR2 protein.
 19. The method of claim18, further comprising administering to the patient from which saidbiological sample was obtained a therapeutically effective amount of atherapeutic agent, wherein the therapeutic agent and/or amount of thetherapeutic agent administered is based upon amount of one or moremodified or unmodified FGFR2 fragment peptides or the level of FGFR2protein.
 20. The method of claim 19, wherein said therapeutic agentbinds the FGFR2 protein and/or inhibits its biological activity.